"Paul W. Diaz" <Pendragn at uclink2.berkeley.edu> wrote:
>I had to make around 50 "linker-scanning" mutagenized promoter
>constructs involving PCR amplication of a 800 bp promoter fragment.
>Only a couple of times did I detect infidelity due to Taq. Curious
>about proofreading thermo-pols as you are, I tried Pfu and found that my
>PCR reactions would work only after substantial troubleshooting (and
>even then, not very well), so I returned to using Taq. You might want
>to give Taq a try, unless the newer generations of proofreading pols
>Our experience has also been that Taq works best with little
optimization. I can't say I've put this to the test but I suspect
that the "long PCR" enzyme mixtures of Taq + a proof reading enzyme
(ala Barnes, PNAS 93 or 94 can't remember for sure) would give reduced
error rate plus easier optimization provided by Taq.
Another point, I haven't tracked down references for this, but someone
told me that some minor contaminants of nucleotides induce high error
rates by polymerases. I vaguely recall that these contaminants are
related to the nucleotide intermediates that accumulate in mutD
strains and are responsible for the high mutagenic rates seen in mutD
strains. Thus, be discriminating about the quality of your
nucleotides critical "no-error" work. And I presume adding these
contaminants could be an efficient method to acheive a controllable
level of mutagensis, if desired.
John R. Thompson
Merck Research Labs